The long-term goal of this research project is to develop strategies for establishing safe, durable engraftment of normal donor stem cells in human fetuses diagnosed with immunodeficiency diseases, hemoglobinopathies or inborn errors of metabolism. In the mouse model of in utero transplantation (IUT), the majority of recipients engraft but at very low levels, insufficient to induce tolerance. This is comparable to what has been seen in monkeys and humans following IUT. We have found that marrow derived dendritic cell progenitors (pDC) added to the marrow inoculum result in virtually full donor hematopoietic chimerism in some animals but at the expense of significant graft versus host disease (GvHD). We believe this is due to the rapid maturation of pDC which then interact with donor T cells. The immediate goal of this research proposal is to understand the mechanisms by which tolerance to allogeneic mismatched hematopoietic stem cells (HSC) can be induced in utero in the fetal mouse. The specific aims of this proposal are: 1) to understand why tolerance is not reliably induced in utero and the role of DC in its induction and 2) to characterize the role of a growth advantage for donor HSC by selectively destroying host hematopoiesis in utero or postnatally. We will utilize mature DC from CD80 and CD86 single and double knockout mice and maturation resistant DC to study the mechanisms for tolerance induction in utero. We will also evaluate the role of "space" by using donor NK cells or sensitized donor CD8+ cells to prepare the recipient for IUT. Finally, we will study the mechanisms of both central and peripheral tolerance in tolerant animals in order to define those that are critical during the early phases of engraftment. The results of these experiments will lead to the development of therapeutic strategies for the induction of tolerance and optimization of engraftment of donor cells pre and postnatally and could have an impact on other areas of transplantation, specifically, induction and maintenance of tolerance to solid organs, and engraftment of HLA-mismatched stem cells post non-myeloablative conditioning.